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Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets

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Abstract

Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for “artificial alpha repeat protein”) have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal “Velcro” to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself.

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Abbreviations

2XTY:

E. coli rich media

A8-35:

A specific type of poly(acrylic acid)-based amphipol

APol:

Amphipol

BAPol:

Biotinylated A8-35

BNAPol:

Biotinylated non-ionic amphipol

BR:

Bacteriorhodopsin

cmc:

Critical micellar concentration

DDM:

Dodecyl-β-d-maltoside

\(\overline{DP}_{n}\) :

Number-average degree of polymerization

EDTA:

Ethylene diamine tetraacetic acid

EM:

Electron microscopy

His-tag:

Hexahistidine tag

IPTG:

Isopropyl-β-d-1-thiogalactopyranoside

MD:

Molecular dynamics

\(\overline{M}_{n}\) :

Number-average molar mass

MP:

Membrane protein

MW:

Molecular weight

MWCO:

MW cut-off

NAPol:

Non-ionic amphipol

NMR:

Nuclear magnetic resonance

NTA:

Nitrilotriacetic acid

OD600nm :

Optical density measured at 600 nm

βOG:

n-octyl-β-d-glucopyranoside

PBS:

Phosphate buffer saline

PEG:

Polyethylene glycol

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEC:

Size-exclusion chromatography

TBS:

Tris-buffered saline

TBST:

Tris-buffered saline supplemented with Tween 20 (w/v)

Tris:

Tris(hydroxymethyl)aminomethane

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Acknowledgments

Particular thanks are due to J.D. Perlmutter and J.N. Sachs for communication of a molecular dynamics model of an A8-35 particle. This research was supported by ANR-2010-BLAN-1535, by the French Centre National de la Recherche Scientifique (CNRS), by Université Paris-5 Paris Descartes, by Université Paris-7 Denis Diderot, and by grant “DYNAMO”, ANR-11-LABX-0011-01 from the French “Initiative d’Excellence” program.

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Authors and Affiliations

  1. Laboratoire de Modélisation et Ingénierie des Protéines, IBBMC UMR 8619, CNRS/Université Paris Sud, 91405, Orsay, France

    Yann Ferrandez, Agathe Urvoas, Marie Valerio-Lepiniec & Philippe Minard

  2. Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie, UMR 8015, CNRS/Université Paris Descartes, Sorbonne Paris Cité, 4 avenue de l’Observatoire, 75270, Paris Cedex 06, France

    Manuela Dezi, Isabelle Broutin & Martin Picard

  3. Equipe Chimie Bioorganique et Systèmes Amphiphiles, Université d’Avignon, 33 rue Louis Pasteur, 84000, Avignon, France

    Mickael Bosco, Grégory Durand & Ange Polidori

  4. Institut des Biomolécules Max Mousseron (UMR 5247), 15 avenue Charles Flahault, 34093, Montpellier Cedex 05, France

    Mickael Bosco, Grégory Durand & Ange Polidori

  5. Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, Institut de Biologie Physico-Chimique (FRC 550), Centre National de la Recherche Scientifique/Université Paris-7, 13, rue Pierre-et-Marie-Curie, 75005, Paris, France

    Christel Le Bon, Fabrice Giusti & Jean-Luc Popot

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  1. Yann Ferrandez
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  2. Manuela Dezi
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  3. Mickael Bosco
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  4. Agathe Urvoas
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  5. Marie Valerio-Lepiniec
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  6. Christel Le Bon
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  7. Fabrice Giusti
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  8. Isabelle Broutin
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  9. Grégory Durand
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  10. Ange Polidori
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  11. Jean-Luc Popot
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  12. Martin Picard
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  13. Philippe Minard
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Correspondence to Martin Picard.

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Ferrandez, Y., Dezi, M., Bosco, M. et al. Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets. J Membrane Biol 247, 925–940 (2014). https://doi.org/10.1007/s00232-014-9707-3

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